Iron is an essential nutrient for virtually every organism, yet it can also be a potent cellular toxin. Dysregulated iron metabolism and iron overload are features of a growing number of human diseases. Some genes involved in cellular iron uptake and export have been identified, yet very little is known about inter- and intracellular iron transport, intracellular iron utilization, and the regulation of these processes. A combination of genetic, biochemical, and cell biological approaches is needed to understand iron metabolism and the role of iron in human disease. These approaches can be combined in the simple eukaryote, Saccharomyces cerevisiae. Studies of metal metabolism in budding yeast have yielded important insights into iron, copper, and zinc metabolism in both humans and pathogenic microorganisms. Genetic studies of iron metabolism in a simple eukaryote will allow us to discover new genes involved in iron homeostasis as well as to determine the cellular response to iron overload and iron deprivation. We have used a variety of strategies to identify genes that are involved in eukaryotic iron homeostasis. Using available genome and protein databases, we have grouped these newly identified genes into families and have begun their functional evaluation. We have identified and genetically characterized a novel system of eukaryotic iron uptake. Four homologous genes regulated as part of the Aft1-regulon (ARN1-4) were found to facilitate the transport of siderophores. We have determined that the ferrichrome (FC) transporter, Arn1, undergoes a distinct pattern of intracellular trafficking in response to ferrichrome. The trafficking of the Arn1p transporter suggested the presence of a high-affinity receptor for FC. Whether this receptor was a separate gene product or part of the Arn1p transporter was unknown. The ARN transporters contain a unique carboxyl-terminal domain consisting of two predicted transmembrane domains, an extracytosolic loop, and a cytosolic tail, which are not present in other MFS transporters. We have conducted a structure-function analysis of this domain to evaluate its role in FC binding and trafficking. Mutations in the extracytosolic loop indicate that it functions as a receptor domain, and the binding of FC to this domain controls the sorting of the transporter. Thus, for this simple eukaryote, FC receptor and transporter functions have been combined in a single gene product. We have further evaluated the role of posttranslational modifications in control of the trafficking of Arn1p. Ubiquitination via the E3 ligase Rsp5 controls multiple steps in the intracellular trafficking of Arn1p, and we are investigating effects of site-specific ubiquitination and the role of ubiquitination on transporter function. Our microarray analysis unexpectedly revealed that other metabolic pathways are regulated in response to iron availability. The iron-dependent enzyme glutamate synthetase is also transcriptionally down-regulated in response to iron deprivation, and we have characterized the transcriptional control regions of this gene to identify the iron-responsive sequences. To identify the transcription factor that controls the iron-dependent activation of GLT1, we have begun a screen for genes that stimulate GLT1 expression under conditions of iron deprivation, and these genes will be evaluated as potential iron-regulated transcription factors. Very little is known about heme transport in eukaryotes and no fungal heme transporters have been identified. We have identified a gene from C. albicans that, when overexpressed in S. cerevisiae, facilitates the uptake of heme. This gene, tentatively termed HUF1 for heme utilization factor 1, is part of a fungal gene family with three homologues in C. albicans and, surprisingly, three orthologues in S. cerevisiae, all localized to the endoplasmic reticulum. Members of this family of genes serve an essential function in yeast, as deletion of two members of this family is lethal in S. cerevisiae. We have obtained genetic and biochemical evidence that the HUF family of genes is involved in cell wall biogenesis and outer chain mannosylation.